Trpv 4 mediates the calcium influx required for flightless-non-muscle myosin interaction and collagen remodeling

Collagen remodeling by phagocytosis requires cell extension formation, which in turn involves interaction of the actin binding protein Flightless I (FliI) with non-muscle myosin IIA (NMMIIA) at cell-matrix adhesion sites. As Ca 2+ plays a central role in controlling actomyosin-dependent functions, we examined how Ca 2+ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca 2+ influx at extensions of fibroblasts. Western Blotting and qPCR showed high expression levels of the Ca 2+ -permeable, Transient Receptor Potential Vanilloid-4 (TRPV4) channel, which coimmunoprecipitated with 1 integrin and localized to adhesions. Blocking antibody to  integrin, the TRPV4-specific antagonist AB159908, or reduction of TRPV4 expression with siRNA, all blocked Ca 2+ influx. These treatments also inhibited interaction of FliI with NMMIIA, reduced the number and length of cell extensions and blocked collagen remodeling. Pull-down assays showed that Ca 2+ depletion inhibited interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed FliINMMIIA interactions require Ca 2+ influx. We conclude that Ca 2+ influx through TRPV4 channels regulates FliI-NMMIIA interactions, which in turn enable generation of the cell extensions essential for collagen remodeling. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le